RNAi in C. elegans is naturally systemic; gene silencing initiated in a single cell or tissue spreads, silencing that gene throughout the animal and its progeny. A C. elegans protein, SID-1, has been identified that is required for the systemic transmission of silencing information. SID- 1 is predicted to contain 11 integral membrane domains and a large extracellular domain suggesting that it may act as a channel or receptor for the uptake or transport of dsRNA. Furthermore, two mammalian genes have been identified with a similar overall predicted structure and extensive homology in the transmembrane domains. A second C. elegans integral membrane protein SID-2 is also required for uptake of silencing information. The goals of this proposal are to investigate the subcellular localization, structure, activity, and regulation of SID-1 and SID-2 and to begin to characterize mammalian SID-1 homologs. To determine whether either SID protein acts as a channel or receptor and to identify and characterize their presumed interactions with dsRNA, their transport activity will be investigated using nematode primary cell lines derived from wild-type and mutant embryos. To corroborate these findings, the transport activity of SID-1 and SID-2 expressed in heterologous systems will be assayed. The mouse SID-1 homologs will be expressed in nematodes and insect cell lines to determine whether they can complement sid-1 mutations or perform any of the functions of SID-1. Mouse embryonic stem cells will be characterized for expression of SID homologs and dsRNA uptake activity as well as cell-tocell spreading of silencing information. Finally, we will begin a functional analysis of the mouse homologs in ES cells and chimera embryos. These studies may have a direct impact on the treatment of human genetic disease and viral infection. RNAi has been shown to effectively inhibit HIV and polio infection and to disable oncogenes, returning cells to normal growth behavior. Although RNAi has been shown to be effective, a major obstacle remains delivery of dsRNA into human cells both in culture and in vivo.